The invention relates to recombinant polypeptides and peptides, which can be used for the diagnosis of tuberculosis. The invention also relates to a process for preparing the above-said polypeptides and peptides, which are in a state of biological purity such that they can be used as part of the active principle in the preparation of vaccines against tuberculosis.
1. Field of the Invention
It also relates to nucleic acids coding for said polypeptides and peptides.
Furthermore, the invention relates to the in vitro diagnostic methods and kits using the above-said polypeptides and peptides and to the vaccines containing the above-said polypeptides and peptides as active principle against tuberculosis.
By xe2x80x9crecombinant polypeptides or peptidesxe2x80x9d it is to be understood that it relates to any molecule having a polypeptidic chain liable to be produced by genetic engineering, through transcription and translation, of a corresponding DNA sequence under the control of appropriate regulation elements within an efficient cellular host. Consequently, the expression xe2x80x9crecombinant polypeptidesxe2x80x9d such as is used herein does not exclude the possibility for the polypeptides to comprise other groups, such as glycosylated groups.
The term xe2x80x9crecombinantxe2x80x9d indeed involves the fact that the polypeptide has been produced by genetic engineering, particularly because it results from the expression in a cellular host of the corresponding nucleic acid sequences which have previously been introduced into the expression vector used in said host.
Nevertheless, it must be understood that this expression does not exclude the possibility for the polypeptide to be produced by a different process, for instance by classical chemical synthesis according to methods used in the protein synthesis or by proteolytic cleavage of larger molecules.
The expression xe2x80x9cbiologically purexe2x80x9d or xe2x80x9cbiological purityxe2x80x9d means on the one hand a grade of purity such that the recombinant polypeptide can be used for the production of vaccinating compositions and on the other hand the absence of contaminants, more particularly of natural contaminants.
2. Description of the Prior Art
Tuberculosis remains a major disease in developing countries. The situation is dramatic in some countries, particularly where high incidence of tuberculosis among AIDS patients represents a new source of dissemination of the disease.
Tuberculosis is a chronic infectious disease in which cell-mediated immune mechanisms play an essential role both for protection against and control of the disease.
Despite BCG vaccination, and some effective drugs, tuberculosis remains a major global problem. Skin testing with tuberculin PPD (protein-purified derivative). largely used for screening of the disease is poorly specific, due to cross reactivity with other pathogenic or environmental saprophytic mycobacteria.
Moreover, tuberculin PPD when used in serological tests (ELISA) does not allow to discriminate between patients who have been vaccinated by BCG, or those who have been primo-infected, from those who are developing evolutive tuberculosis and for whom an early and rapid diagnosis would be necessary.
A protein with a molecular weight of 32-kDa has been purified (9) from zinc deficient Mycobacterium bovis BCG culture filtrate (8). This 32-kDa protein of M. bovis BCG has been purified from Sauton zinc deficient culture filtrate of M. bovis BCG using successively hydrophobic chromatography on Phenyl-Sepharose, ion exchange on DEAE-Sephacel and molecular sieving on Sephadex G-lO0. The final preparation has been found to be homogeneous as based on several analyses. This P32 protein is a constituent of BCG cells grown in normal conditions. It represents about 3% of the soluble fraction of a cellular extract, and appears as the major protein released in normal Sauton culture filtrate. This protein has been found to have a molecular weight of 32000 by SDS-polyacrylamide gel electrophoresis and by molecular sieving.
The NH2-terminal amino acid sequence of the 32-kDa protein of M. bovis BCG (Phe-Ser-Arg-Pro-Gly-Leu) is identical to that reported for the MPB 59 protein purified from M. bovis BCG substrain Tokyo (34).
Purified P32 of M. bovis BCG has been tested by various cross immunoelectrophoresis techniques, and has been shown to belong to the antigen 85 complex in the reference system for BCG antigens. It has been more precisely identified as antigen 85A in the Closs reference system for BCG antigens (7).
Increased levels of immunoglobulin G antibodies towards the 32-kDa protein of M. bovis BCG could be detected in 70% of tuberculous patients (30).
Furthermore, the 32-kDa protein of M. bovis BCG induces specific lymphoproliferation and interferon-(IFN-xcex3) production in peripheral blood leucocytes from patients with active tuberculosis (12) and PPD-positive healthy subjects. Recent findings indicate that the amount of 32-kDa protein of M. bovis BCG-induced IFN-xcex3 in BCG-sensitized mouse spleen cells is under probable H-2 control (13). Finally, the high affinity of mycobacteria for fibronectin is related to proteins of the BCG 85 antigen complex (1).
Matsuo et al. (17) recently cloned the gene encoding the antigen xcex1, a major protein secreted by BCG (substrain Tokyo) and highly homologous to MPB 59 antigen in its NH2-terminal amino acid sequence, and even identical for its first 6 amino acids: Phe-Ser-Arg-Pro-Gly-Leu.
This gene was cloned by using a nucleotide probe homologous to the N-terminal amino acid sequence of antigen xcex1, purified from M. tuberculosis as described in Tasaka, H. et al., 1983. xe2x80x9cPurification and antigenic specificity of alpha protein (Yoneda and Fukui) from Mycobacterium tuberculosis and Mycobacterium intracellulare. Hiroshima J. Med. Sci. 32, 1-8.
The presence of antigens of around 30-32-kDa, named antigen 85 complex, has been revealed from electrophoretic patterns of proteins originating from culture media of mycobacteria, such as Mycobacterium tuberculosis. By immunoblotting techniques, it has been shown that these antigens cross-react with rabbit sera raised against the 32-kDa protein of BCG (8).
A recent study reported on the preferential humoral response to a 30-kDa and 31-kDa antigen in lepromatous leprosy patients, and to a 32-kDa antigen in tuberculoid leprosy patients (24).
It has also been found that fibronectin (FN)-binding antigens are prominent components of short-term culture supernatants of Mycobacterium tuberculosis. In 3-day-old supernatants, a 30-kilodalton (kDa) protein was identified as the major (FN)-binding molecule. In 21-day-old supernatants, FN was bound to a double protein band of around 30 to 32-kDa, as well as to a group of antigens of larger molecular mass (57 to 60 kDa) (1).
In other experiments, recombinant plasmids containing DNA from Mycobacterium tuberculosis were transformed into Escherichia coli, and three colonies were selected by their reactivity with polyclonal antisera to M. tuberculosis. Each recombinant produced 35- and 53-kilodalton proteins (35K and 53K proteins, respectively)(xe2x80x9cExpression of Proteins of Mycobacterium tuberculosis in Escherichia coli and Potential of Recombinant Genes and Proteins for Development of Diagnostic Reagentsxe2x80x9d, Mitchell L Cohen et al., Journal of Clinical Microbiology, July 1987, p.1176-1180).
Concerning the various results known to date, the physico-chemical characteristics of the antigen P32 of Mycobacterium tuberculosis are not precise and, furthermore, insufficient to enable its unambiguous identifiability, as well as the characterization of its structural and functional elements.
Moreover, the pathogenicity and the potentially infectious property of M. tuberculosis has hampered research enabling to identify, purify and characterize the constituents as well as the secretion products of this bacteria.
An aspect of the invention is to provide recombinant polypeptides which can be used as purified antigens for the detection and control of tuberculosis.
Another aspect of the invention is to provide nucleic acids coding for the peptidic chains of biologically pure recombinant polypeptides which enable their preparation on a large scale.
Another aspect of the invention is to provide antigens which can be used in serological tests as an in vitro rapid diagnostic of tuberculosis.
Another aspect of the invention is to provide a rapid in vitro diagnostic means for tuberculosis, enabling it to discriminate between patients suffering from an evolutive tuberculosis from those who have been vaccinated against BCG or who have been primo-infected.
Another aspect of the invention is to provide nucleic probes which can be used as in vitro diagnostic reagent for tuberculosis, as well as in vitro diagnostic reagent for identifying M. tuberculosis from other strains of mycobacteria.
The recombinant polypeptides of the invention contain in their polypeptidic chain one at least of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (xe2x88x9229) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (12) to the extremity constituted by amino acid at position (31) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (36) to the extremity constituted by amino acid at position (55) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (77) to the extremity constituted by amino acid at position (96) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (101) to the extremity constituted by amino acid at position (120) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (175) to the extremity constituted by amino acid at position (194) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (211) to the extremity constituted by amino acid at position (230) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (275) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
and the peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids in so far as this modification does not alter the following properties:
the polypeptides react with rabbit polyclonal antiserum raised against the protein of 32-kDa of M. bovis BCG culture filtrate, and/or
react selectively with human sera from tuberculosis patients and particularly patients developing an evolutive tuberculosis at an early stage,
and/or react with the amino acid sequence extending from the extremity constituted by amino acid at position (1), to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b. 
On FIGS. 3a and 3b: 
X represents G or GG,
Y represents C or CC,
Z represents C or G,
W represents C or G and is different from Z,
K represents C or CG,
L represents G or CC,
a1-b1 represents ALA-ARG or GLY-ALA-ALA,
a2 represents arg or gly,
a3-b3-c3-d3-e3-f3-represents his-trp-val-pro-arg-pro or ala-leu-gly-ala,
a4 represents pro or pro-asn-thr,
a5 represents pro or ala-pro.
The recombinant polypeptides of the invention contain in their polypeptidic chain one at least of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (xe2x88x9229) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (12) to the extremity constituted by amino acid at position (31) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (36) to the extremity constituted by amino acid at position (55) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (77) to the extremity constituted by amino acid at position (96) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (101) to the extremity constituted by amino acid at position (120) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (175) to the extremity constituted by amino acid at position (194) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (211) to the extremity constituted by amino acid at position (230) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (275) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
and the peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids in so far as this modification does not alter the following properties:
the polypeptides react with rabbit polyclonal antiserum raised against the protein of 32-kDa of M. bovis BCG culture filtrate, and/or
react selectively with human sera from tuberculosis patients and particularly patients developing an evolutive tuberculosis at an early stage,
and/or react with the amino acid sequence extending from the extremity constituted by amino acid at position (1), to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b. 
The recombinant polypeptides of the invention contain in their polypeptidic chain one at least of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (xe2x88x9230) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (12) to the extremity constituted by amino acid at position (31) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (36) to the extremity constituted by amino acid at position (55) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (77) to the extremity constituted by amino acid at position (96) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (101) to the extremity constituted by amino acid at position (120) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (175) to the extremity constituted by amino acid at position (194) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (211) to the extremity constituted by amino acid at position (230) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (275) to the extremity constituted by amino acid at position (295) represented on FIG. 5,
and the peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids in so far as this modification does not alter the following properties:
the polypeptides react with rabbit polyclonal antiserum raised against the protein of 32-kDa of M. bovis BCG culture filtrate, and/or
react selectively with human sera from tuberculosis patients and particularly patients developing an evolutive tuberculosis at an early stage,
and/or react with the amino acid sequence extending from the extremity constituted by amino acid at position (1), to the extremity constituted by amino acid at position (295) represented on FIG. 5.
Advantageous polypeptides of the invention are characterized by the fact that they react with rabbit polyclonal antiserum raised against the protein of 32-kDa of M. bovis BCG culture filtrate, hereafter designated by xe2x80x9cP32 protein of BCGxe2x80x9d.
Advantageous polypeptides of the invention are characterized by the fact that they selectively react with human sera from tuberculous patients and particularly patients developing an evolutive tuberculosis at an early stage.
Hereafter is given, in a non limitative way a process for preparing rabbit polyclonal antiserum raised against the P32 protein of BCG and a test for giving evidence of the reaction between the polypeptides of the invention and said rabbit polyclonal antiserum raised against the P32 protein of BCG.
1) Process for Preparing Rabbit Polyclonal Antiserum Raised Against the P32 Protein of BCG:
Purified P32 protein of BCG from culture filtrate is used.
a) Purification of Protein P32 of BCG:
P32 protein can be purified as follows:
The bacterial strains used are M. bovis BCG substrains 1173P2 (Pasteur Institute, Paris) and GL2 (Pasteur Institute, Brussels).
The culture of bacteria is obtained as follows
Mycobacterium bovis BCG is grown as a pellicle on Sauton medium;
containing 4 g Aspargine, 57 ml 99% Glycerine (or 60 ml 87% Glycerine), 2 g Citric Acid, 0.5 g K2HPO4, 0.5 g MgSO4, 0.05 g Citrate, 5xc3x9710xe2x88x926 M Ammonium (17% Fe III) SO4Zn-7H2O and adjusted to 1 liter distilled water adjusted to pH 7.2 with NH4OH. at 37.5xc2x0 C. for 14 days. As the medium is prepared with distilled water, zinc sulfate is added to the final concentration of 5 xcexcM (normal Sauton medium)(De Bruyn J., Weckx M., Beumer-Jochmans M.-P. Effect of zinc deficiency on Mycobacterium tuberculosis var. bovis (BCG). J. Gen. Microbiol. 1981; 124:353-7). When zinc deficient medium was needed, zinc sulfate is omitted.
The filtrates from zinc deficient cultures are obtained as follows:
The culture medium is clarified by decantation. The remaining bacteria are removed by filtration through Millipak 100 filter unit (Millipore Corp., Bedford, Mass.). When used for purification, the filtrate is adjusted to 20 mM in phosphate, 450 mM. in NaCl, 1 mM in EDTA, and the pH is brought to 7.3 with 5 M HCl before sterile filtration.
The protein analysis is carried out by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was done on 13% (w/v) acrylamide-containing gels as described by Laemmli UK. (Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-5). The gels are stained with Coomassie Brilliant Blue R-250 and for quantitative analysis, scanned at 595 nm with a DU8 Beckman spectrophotometer. For control of purity the gel is revealed with silver stain (Biorad Laboratories, Richmond, Calif.).
The purification step of P32 is carried out as follows:
Except for hydrophobic chromatography on Phenyl-Sepharose, all buffers contain Tween 80 (0.005% final concentration). The pH is adjusted to 7.3 before sterilization. All purification steps are carried out at +4xc2x0 C. Elutions are followed by recording the absorbance at 280 nm. The fractions containing proteins are analysed by SDS-PAGE.
(i) The treated filtrate from a 4 liters zinc-deficient culture, usually containing 125 to 150 mg protein per liter, is applied to a column (5.0 by 5.0 cm) of Phenyl-Sepharose CL-4B (Pharmacia Fine Chemicals, Uppsala, Sweden), which is previously equilibrated with 20 mM phosphate buffer (PB) containing 0.45 M NaCl and 1 mM EDTA, at a flow rate of 800 ml per hour. The gel is then washed with one column volume of the same buffer to remove unfixed material and successively with 300 ml of 20 mM and 4 mM PB and 10% ethanol (v/v). The P32 appears in the fraction eluted with 10% ethanol.
(ii) After the phosphate concentration of this fraction has been brought to 4 mM, it is applied to a column (2.6 by 10 cm) of DEAE-Sephacel (Pharmacia Fine Chemicals), which is equilibrated with 4 mM PB. After washing with the equilibrating buffer the sample is eluted with 25 mM phosphate at a flow rate of 50 ml per hour. The eluate is concentrated in a 202 Amicon stirred cell equipped with a PM 10 membrane (Amicon Corp., Lexington, Mass.).
(iii) The concentrated material is submitted to molecular sieving on a Sephadex G-100 (Pharmacia) column (2.6 by 45 cm) equilibrated with 50 mM PB, at a flow rate of 12 ml per hour. The fractions of the peak giving one band in SDS-PAGE are pooled. The purity of the final preparation obtained is controlled by SDS-PAGE followed by silverstaining and by molecular sieving on a Superose 12 (Pharmacia) column (12.0 by 30 cm) equilibrated with 50 mM PB containing 0.005% Tween 80 at a flow rate of 0.2 ml/min. in the Fast Protein Liquid Chromatography system (Pharmacia). Elution is followed by recording the absorbance at 280 nm and 214 nm.
b) Preparation of Rabbit Polyclonal Antiserum Raised Against the P32 Protein of BCG
400 xcexcg of purified P32 protein of BCG per ml physiological saline are mixed with one volume of incomplete Freund""s adjuvant. The material is homogenized and injected intradermally in 50 xcexcl doses delivered at 10 sites in the back of the rabbits, at 0, 4, 7 and 8 weeks (adjuvant is replaced by the diluent for the last injection). One week later, the rabbits are bled and the sera tested for antibody level before being distributed in aliquots and stored at xe2x88x9280xc2x0 C.;
2) Test for Giving Evidence of the Reaction Between the Polypeptides of the Invention and Said Rabbit Polyclonal Antiserum Raised Against the P32 Protein of BCG:
The test used was an ELISA test; the ELISA for antibody determination is based on the method of Enqvall and Perlmann (Engvall, E., and P. Perlmann. 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871-874)
Immulon Microelisa plates (Dynatech, Kloten, Switzerland) are coated by adding to each well 1 xcexcg of one of the polypeptides of the invention in 100 xcexcl Tris hydrochloride buffer 50 mM (pH 8.2). After incubation for 2 h at 27xc2x0 C. in a moist chamber, the plates are kept overnight at 4xc2x0 C. They are washed four times with 0.01 M phosphate-buffered saline (pH 7.2) containing 0.05% Tween 20 by using a Titertek microplate washer (Flow Laboratories. Brussels. Belgium). Blocking is done with 0.5% gelatin in 0.06 M carbonate buffer (pH 9.6) for 1 h. Wells are then washed as before, and 100 xcexcl of above mentioned serum diluted in phosphate-buffered saline containing 0.05% Tween 20 and 0.5% gelatin is added. According to the results obtained in preliminary experiments, the working dilutions are set at 1:200 for IgG, 1:20 for IgA and 1:80 for IgM determinations. Each dilution is run in duplicate. After 2 h of incubation and after the wells are washed, they are filled with 100 xcexcl of peroxidase-conjugated rabbit immunoglobulins directed against human IgG, IgA or IgM (Dakopatts, Copenhagen, Denmark), diluted 1:400, 1:400 and 1:1.200, respectively in phosphate-buffered saline containing 0.05% Tween 20 and 0.5% gelatin and incubated for 90 min. After the wash, the amount of peroxidase bound to the wells is quantified by using a freshly prepared solution of o-phenylenediamine (10 mg/100 ml) and hydrogen peroxide (8 xcexcl of 30% H2O2 per 100 ml) in 0.15 M citrate buffer (pH 5.0) as a substrate. The enzymatic reaction is stopped with 8 N H2SO4 after 15 min. of incubation. The optical density is read at 492 nm with a Titertek Multiskan photometer (Flow Laboratories).
Wells without sera are used as controls for the conjugates. Each experiment is done by including on each plate one negative and two positive reference sera with medium and low antibody levels to correct for plate-to-plate and day-to-day variations. The antibody concentrations are expressed as the optical density values obtained after correction of the readings according to the mean variations of the reference sera.
Hereafter is also given in a non limitative way, a test for giving evidence of the fact that polypeptides of the invention are recognized selectively by human sera from tuberculous patients.
This test is an immunoblotting (Western blotting) analysis, in the case where the polypeptides of the invention are obtained by recombinant techniques. This test can also be used for polypeptides of the invention obtained by a different preparation process. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polypeptides of the invention are blotted onto nitrocellulose membranes (Hybond C. (Amersham)) as described by Towbin et al. (29). The expression of polypeptides of the invention fused to xcex2-galactosidase in E. coli Y1089, is visualized by the binding of a polyclonal rabbit anti-32-kDa BCG protein serum (1:1,000) or by using a monoclonal anti-xcex2-galactosidase antibody (Promega). The secondary antibody (alkaline phosphatase anti-rabbit immunoglobulin G and anti-mouse alkaline phosphatase immunoglobulin G conjugates, respectively) is diluted as recommended by the supplier (Promega).
In order to identify selective recognition of polypeptides of the invention and of fusion proteins of the invention by human tuberculous sera, nitrocellulose sheets are incubated overnight with these sera (1:50) (after blocking aspecific protein-binding sites). The human tuberculous sera are selected for their reactivity (high or low) against the purified 32-kDa antigen of BCG tested in a dot blot assay as described in document (31) of the bibliography hereafter. Reactive areas on the nitrocellulose sheets are revealed by incubation with peroxidase conjugated goat anti-human immunoglobulin G antibody (Dakopatts, Copenhagen, Denmark)(1:200) for 4 h, and after repeated washings, color reaction is developed by adding peroxidase substrate (xcex1-chloronaphtol)(Bio-Rad Laboratories, Richmond, Calif.) in the presence of peroxidase and hydrogen peroxide.
It goes without saying that the free reactive functions which are present in some of the amino acids, which are part of the constitution of the polypeptides of the invention, particularly the free carboxyl groups which are carried by the groups Glu or by the C-terminal amino acid on the one hand and/or the free NH2 groups carried by the N-terminal amino acid or by amino acid inside the peptidic chain, for instance Lys, on the other hand, can be modified in so far as this modification does not alter the above mentioned properties of the polypeptide.
The molecules which are thus modified are naturally part of the invention. The above mentioned carboxyl groups can be acylated or esterified.
Other modifications are also part of the invention. Particularly, the amine or ester functions or both of terminal amino acids can be themselves involved in the bond with other amino acids. For instance, the N-terminal amino acid can be linked to a sequence comprising from 1 to several amino acids corresponding to a part of the C-terminal region of another peptide.
Furthermore, any peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids of the polypeptides according to the invention are part of the invention in so far as this modification does not alter the above mentioned properties of said polypeptides.
The polypeptides according to the invention can be glycosylated or not, particularly in some of their glycosylation sites of the type Asn-X-Ser or Asn-X-Thr, X representing any amino acid.
Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (xe2x88x9242) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9247) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9249) to to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9255) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9259) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 3a and FIG. 3b. 
Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (xe2x88x9242) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9247) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9249) to to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9255) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9259) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 4a and FIG. 4b. 
Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (xe2x88x9243) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 5.
Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9229) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9242) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9247) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9249) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9255) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9259) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b. 
Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9229) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9242) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9247) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9249) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9255) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9259) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b. 
Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (295) represented on FIG. 5,
the one extending from the extremity constituted by amino acid at position (xe2x88x9230) to the extremity constituted by amino acid at position (295) represented on FIG. 5,
the one extending from the extremity constituted by amino acid at position (xe2x88x9243) to the extremity constituted by amino acid at position (295) represented on FIG. 5.
Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (xe2x88x9259) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9255) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9249) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9247) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9242) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9229) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b. 
Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (xe2x88x9259) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9255) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9249) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9247) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9242) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9229) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b. 
Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (295) represented on FIG. 5,
the one extending from the extremity constituted by amino acid at position (xe2x88x9230) to the extremity constituted by amino acid at position (295) represented on FIG. 5,
the one extending from the extremity constituted by amino acid at position (xe2x88x9243) to the extremity constituted by amino acid at position (295) represented on FIG. 5.
Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (xe2x88x9259) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9255) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9249) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9247) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9242) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9229) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 3a and FIG. 3b. 
Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (xe2x88x9259) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9255) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9249) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9247) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9242) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by amino acid at position (xe2x88x9229) to the extremity constituted by amino acid at position (xe2x88x921) represented aon FIG. 4a and FIG. 4b. 
Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (xe2x88x9243) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 5,
the one extending from the extremity constituted by amino acid at position (xe2x88x9230) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 5.
In eukaryotic cells, these polypeptides can be used as signal peptides, the role of which is to initiate the translocation of a protein from its site of synthesis, but which is excised during translocation.
Other advantageous peptides of the invention consist in one of the following amino acid sequence:
the one extending from the extremity constituted by amino acid at position (12) to the extremity constituted by amino acid at position (31) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (36) to the extremity constituted by amino acid at position (55) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (77) to the extremity constituted by amino acid at position (96) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (101) to the extremity constituted by amino acid at position (120) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (175) to the extremity constituted by amino acid at position (194) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (211) to the extremity constituted by amino acid at position (230) represented on FIG. 3a and FIG. 3b, or
the one extending from the extremity constituted by amino acid at position (275) to the extremity constituted by amino acid at position (294) represented on FIG. 3a and FIG. 3b. 
Other advantageous peptides of the invention consist in one of the following amino acid sequence:
the one extending from the extremity constituted by amino acid at position (12) to the extremity constituted by amino acid at position (31) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (36) to the extremity constituted by amino acid at position (55) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (77) to the extremity constituted by amino acid at position (96) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (101) to the extremity constituted by amino acid at position (120) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (175) to the extremity constituted by amino acid at position (194) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (211) to the extremity constituted by amino acid at position (230) represented on FIG. 4a and FIG. 4b, or
the one extending from the extremity constituted by amino acid at position (275) to the extremity constituted by amino acid at position (294) represented on FIG. 4a and FIG. 4b. 
Other advantageous peptides of the invention consist in one of the following amino acid sequence:
the one extending from the extremity constituted by amino acid at position (12) to the extremity constituted by amino acid at position (31) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (36) to the extremity constituted by amino acid at position (55) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (77) to the extremity constituted by amino acid at position (96) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (101) to the extremity constituted by amino acid at position (120) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (175) to the extremity constituted by amino acid at position (194) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (211) to the extremity constituted by amino acid at position (230) represented on FIG. 5, or
the one extending from the extremity constituted by amino acid at position (275) to the extremity constituted by amino acid at position (295) represented on FIG. 5.
It is to be noted that the above mentioned polypeptides are derived from the expression products of a DNA derived from the nucleotide sequence coding for a protein of 32-kDa secreted by Mycobacterium tuberculosis as explained hereafter in the examples.
The invention also relates to the amino acid sequences constituted by the above mentioned polypeptides and a protein or an heterologous sequence with respect to said polypeptide, said protein or heterologous sequence comprising for instance from about 1 to about 1000 amino acids. These amino acid sequences will be called fusion proteins.
In an advantageous fusion protein of the invention, the heterologous protein is xcex2-galactosidase.
Other advantageous fusion proteins of the invention are the ones containing an heterologous protein resulting from the expression of one of the following plasmids:
pEX1
pEX2
pEX3
pUEX1 pmTNF MPH
pUEX2
pUEX3
The invention also relates to any nucleotide sequence coding for a polypeptide of the invention.
The invention also relates to nucleic acids comprising nucleotide sequences which hybridize with the nucleotide sequences coding for any of the above mentioned polypeptides under the following hybridization conditions:
hybridization and wash medium: 3xc3x97SSC, 20% formamide (1xc3x97SSC is 0,15 M NaCl, 0.015 M sodium citrate, pH 7.0),
hybridization temperature (HT) and wash temperature (WT) for the nucleic acids of the invention defined by x-y: i.e. by the sequence extending from the extremity consituted by the nucleotide at position (x) to the extremity constituted by the nucleotide at position (y) represented on FIG. 3a and FIG. 3b. 
1-182 HT=WT=69xc2x0 C.
1-194 HT=WT=69xc2x0 C.
1-212 HT=WT=69xc2x0 C.
1-218 HT=WT=69xc2x0 C.
1-272 HT=WT=69xc2x0 C.
1-359 HT=WT=71xc2x0 C.
1-1241 HT=WT=73xc2x0 C.
1-1358 HT=WT=73xc2x0 C.
183-359 HT=WT=70xc2x0 C.
183-1241 HT=WT=73xc2x0 C.
183-1358 HT=WT=73xc2x0 C.
195-359 HT=WT=70xc2x0 C.
195-1241 HT=WT=73xc2x0 C.
195-1358 HT=WT=73xc2x0 C.
213-359 HT=WT=70xc2x0 C.
213-1241 HT=WT=73xc2x0 C.
213-1358 HT=WT=73xc2x0 C.
219-359 HT=WT=71xc2x0 C.
219-1241 HT=WT=73xc2x0 C.
219-1358 HT=WT=73xc2x0 C.
234-359 HT=WT=71xc2x0 C.
234-1241 HT=WT=74xc2x0 C.
234-1358 HT=WT=73xc2x0 C.
273-359 HT=WT=71xc2x0 C.
273-1241 HT=WT=74xc2x0 C.
273-1358 HT=WT=73xc2x0 C.
360-1241 HT=WT=73xc2x0 C.
360-1358 HT=WT=73xc2x0 C.
1242-1358 HT=WT=62xc2x0 C.
The above mentioned temperatures are to be considered as approximately xc2x15xc2x0 C.
The invention also relates to nucleic acids comprising nucleotide sequences which are complementary to the nucleotide sequences coding for any of the above mentioned polypeptides.
It is to be noted that in the above defined nucleic acids, as well as in the hereafter defined nucleic acids, the nucleotide sequences which are brought into play are such that T can be replaced by U.
A group of preferred nucleic acids of the invention comprises one at least of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (182) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1242) to the extremity constituted by nucleotide at position (1358), wherein N represents one of the five A, T, C, G or I nucleotides, represented in FIG. 3a and FIG. 3b, 
or above said nucleotide sequences wherein T is replaced by U,
or nucleic acids which hybridize with said above nmentioned nucleotide sequences or the complementary sequences thereof.
A group of preferred nucleic acids of the invention comprises one at least of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (182) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1242) to the extremity constituted by nucleotide at position (1358), wherein N represents one of the five A, T, C, G or I nucleotides, represented in FIG. 4a and FIG. 4b, 
or above said nucleotide sequences wherein T is replaced by U,
or nucleic acids which hybridize with said above mentioned nucleotide sequences or the complementary sequences thereof.
A group of preferred nucleic acids of the invention comprises one at least of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (130) to the extremity constituted by nucleotide at position (219) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (220) to the extremity constituted by nucleotide at position (1104) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (1104) to the extremity constituted by nucleotide at position (1299), wherein N represents one of the five A, T, C, G or I nucleotides, represented in FIG. 5,
or above said nucleotide sequences wherein T is replaced by U,
or nucleic acids which hybridize with said above mentioned nucleotide sequences or the complementary sequences thereof.
Other preferred nucleic acids of the invention comprise one at least of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b. 
Other preferred nucleic acids of the invention comprise one at least of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b. 
Another preferred group of nucleic acids of the invention comprises the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1358) represented, in FIG. 3a and FIG. 3b. 
Another preferred group of nucleic acids of the invention comprises the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b. 
According to another advantageous embodiment, nucleic acids of the invention comprises one of the following sequences:
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (194) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (212) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (218) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (272) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b. 
According to another advantageous embodiment, nucleic acids of the invention comprises one of the following sequences:
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (194) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (212) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (218) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (272) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1241represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b. 
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b. 
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b. 
These nucleotide sequence can be used as nucleotide signal sequences, coding for the corresponding signal peptide.
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b. 
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (182) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (194) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (212) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (218) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (272) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (359) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1241) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b, 
the one extending from the extremity constituted by nucleotide at position (1242) to the extremity constituted by nucleotide at position (1358) represented in FIG. 3a and FIG. 3b. 
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (360) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b. 
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (182) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (194) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (212) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (218) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (272) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (359) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (183) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (195) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (213) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (219) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (234) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1241) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (273) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b, 
the one extending from the extremity constituted by nucleotide at position (1242) to the extremity constituted by nucleotide at position (1358) represented in FIG. 4a and FIG. 4b. 
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (129) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (219) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1104) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1299) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (90) to the extremity constituted by nucleotide at position (219) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (90) to the extremity constituted by nucleotide at position (1299) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (90) to the extremity constituted by nucleotide at position (1104) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (130) to the extremity constituted by nucleotide at position (1104) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (130) to the extremity constituted by nucleotide at position (1299) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (220) to the extremity constituted by nucleotide at position (1299) represented in FIG. 5.
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (129) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (219) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1104) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (1) to the extremity constituted by nucleotide at position (1299) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (90) to the extremity constituted by nucleotide at position (219) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (90) to the extremity constituted by nucleotide at position (1104) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (90) to the extremity constituted by nucleotide at position (1299) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (130) to the extremity constituted by nucleotide at position (219) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (130) to the extremity constituted by nucleotide at position (1104) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (130) to the extremity constituted by nucleotide at position (1299) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (220) to the extremity constituted by nucleotide at position (1104) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (220) to the extremity constituted by nucleotide at position (1299) represented in FIG. 5,
the one extending from the extremity constituted by nucleotide at position (1104) to the extremity constituted by nucleotide at position (1299) represented in FIG. 5.
The invention also relates to any recombinant nucleic acids containing at least a nucleic acid of the invention inserted in an heterologous nucleic acid.
The invention relates more particularly to recombinant nucleic acid such as defined, in which the nucleotide sequence of the invention is preceded by a promoter (particularly an inducible promoter) under the control of which the transcription of said sequence is liable to be processed and possibly followed by a sequence coding for transcription termination signals.
The invention also relates to the recombinant nucleic acids in which the nucleic acid sequences coding for the polypeptide of the invention and possibly the signal peptide, are recombined with control elements which are heterologous with respect to the ones to which they are normally associated within the bacteria gene and, more particularly, the regulation elements adapted to control their expression in the cellular host which has been chosen for their production.
The invention also relates to recombinant vectors, particularly for cloning and/or expression, comprising a vector sequence, notably of the type plasmid, cosmid or phage, and a recombinant nucleic acid of the invention, in one of the non essential sites for its replication.
Appropriate vectors for expression of the recombinant antigen are the following one:
pEX1 pmTNF MPH
pEX2 pIGRI
pEX3
pUEX1
pUEX2
pUEX3
The pEX1, pEX2 and pEX3 vectors are commercially available and can be obtained from Boehringer Mannheim.
The pUEX1, pUEX2 and pUEX3 vectors are also commercially available and can be obtained from Amersham.
According to an advantageous embodiment of the invention, the recombinant vector contains, in one of its non essential sites for its replication, necessary elements to promote the expression of polypeptides according to the invention in a cellular host and possibly a promoter recognized by the polymerase of the cellular host, particularly an inducible promoter and possibly a signal sequence and/or an anchor sequence.
According to another additional embodiment of the invention, the recombinant vector contains the elements enabling the expression by E. coli of a nucleic acid according to the invention inserted in the vector, and particularly the elements enabling the expression of the gene or part thereof of xcex2-galactosidase.
The invention also relates to a cellular host which is transformed by a recombinant vector according to the invention, and comprising the regulation elements enabling the expression of the nucleotide sequence coding for the polypeptide according to the invention in this host.
The invention also relates to a cellular host chosen from among bacteria such as E. coli, transformed by a vector as above defined, and defined hereafter in the examples, or chosen from among eukaryotic organism, such as CHO cells, insect cells, Sf9 cells [Spodoptera frugiperda] infected by the virus Ac NPV (Autographa californica nuclear polyhydrosis virus) containing suitable vectors such as pAc 373 pYM1 or pVC3, BmN [Bombyx mori] infected by the virus BmNPV containing suitable vectors such as pBE520 or p89B310.
The invention relates to an expression product of a nucleic acid expressed by a transformed cellular host according to the invention.
The invention also relates to nucleotidic probes, hybridizing with anyone of the nucleic acids or with their complementary sequences, and particularly the probes chosen among the following nucleotidic sequences gathered in Table 1, and represented in FIG. 9.
Probes A(i), A(ii), A(iii), A(iv) and A(v)
A(i) CAGCTTGTTGACAGGGTTCGTGGC
A(ii) GGTTCGTGGCGCCGTCACG
A(iii) CGTCGCGCGCCTAGTGTCGG
A(iv) CGGCGCCGTCGGTGGCACGGCGA
A(v) CGTCGGCGCGGCCCTAGTGTCGG
Probe B
TCGCCCGCCCTGTACCTG
Probe C
GCGCTGACGCTGGCGATCTATC
Probe D
CCGCTGTTGAACGTCGGGAAG
Probe E
AAGCCGTCGGATCTGGGTGGCAAC
Probes F(i), F(ii), F(iii) and F(iv)
F(i) ACGGCACTGGGTGCCACGCCCAAC
F(ii) ACGCCCAACACCGGGCCCGCCGCA
F(iii) ACGGGCACTGGGTGCCACGCCCAAC
F(iv) ACGCCCCAACACCGGGCCCGCGCCCCA
or their complementary nucleotidic sequences.
The hybridization conditions can be the following ones:
hybridization and wash medium: 3xc3x97SSC, 20% formamide (1xc3x97SSC is 0,15 M NaCl, 0.015 M sodium citrate, pH 7.0),
hybridization temperature (HT) and wash temperature (WT):
These probes might enable to differentiate M. tuberculosis from other bacterial strains and in particular from the following mycobacteria species:
Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium gordonae, Mycobacterium szulgai, Mycobacterium intracellulare, Mycobacterium xenopi, Mycobacterium gastri, Mycobacterium nonchromogenicum, Mycobacterium terrae and Mycobacterium triviale, and more particularly from M. bovis, Mycobacterium kansasii, Mycobacterium avium, Mycobacterium phlei and Mycobacterium fortuitum. 
The invention also relates to DNA or RNA primers which can be used for the synthesis of nucleotidic sequences according to the invention by PCR (polymerase chain reaction technique), such as described in U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,683,195 and European Patent No. 200362.
The invention also relates to any DNA or RNA primer constituted by about 15 to about 25 nucleotides of a nucleotide sequence coding for a polypeptide according to the invention.
The invention also relates to any DNA or RNA primer constituted by about 15 to about 25 nucleotides liable to hybridize with a nucleotide sequence coding for a polypeptide according to the invention.
The invention also relates to any DNA or RNA primer constituted by about 15 to about 25 nucleotides complementary to a nucleotide sequence coding for a polypeptide according to the invention.
The sequences which can be used as primers are given in Table 2 hereafter (sequences P1 to P6 or their complement) and illustrated in FIG. 9:
The sequences can be combined in twelve different primer-sets (given in Table 3) which allow enzymatical amplification by the polymerase chain reaction (PCR) technique of any of the nucleotide sequences of the invention, and more particularly the one extending from the extremity constituted by nucleotide at position 1 to the extremity constituted by nucleotide at position 1358, as well as the nucleotide sequence of antigen xcex1 of BCG (17).
The detection of the PCR amplified product can be achieved by a hybridization reaction with an oligonucleotide sequence of at least 10 nucleotides which is located between PCR primers which have been used to amplify the DNA.
The PCR products of the nucleotide sequences of the invention can be distinguished from the xcex1-antigen gene of BCG or part thereof by hybridization techniques (dot-spot, Southern blotting, etc.) with the probes indicated in Table 3. The sequences of these probes can be found in Table 1 hereabove.
It is to be noted that enzymatic amplification can also be achieved with all oligonucleotides with sequences of about 15 consecutive bases of the primers given in Table 2. Primers with elongation at the 5xe2x80x2-end or with a small degree of mismatch may not considerably affect the outcome of the enzymatic amplification if the mismatches do not interfere with the base-pairing at the 3xe2x80x2-end of the primers.
Specific enzymatic amplification of the nucleotide sequences of the invention and not of the BCG gene can be achieved when the probes (given in Table 1) or their complements are used as amplification primers.
When the above mentioned probes of Table 1 are used as primers, the primer sets are constituted by any of the nucleotide sequences (A, B, C, D, E, F) of Table 1 in association with the complement of any other nucleotide sequence, chosen from A, B, C, D, E or F, it being understood that sequence A means any of the sequences A(i), A(ii), A(iii), A(iv), A(v) and sequence F, any of the sequences F(i), F(ii), F(iii) and F(iv).
Advantageous primer sets for enzymatic amplification of the nucleotide sequence of the invention can be one of the following primer sets given in Table 3bis hereafter:
A(i)
or A(ii)
or A(iii) and the complement of B
or A(iv)
or A(v)
A(i)
or A(ii)
or A(iii) and the complement of C
or A(iv)
or A(v)
B and the complement of C
A(i)
or A(ii)
or A(iii) and the complement of F
or A(iv)
or A(v)
A(i)
or A(ii)
or A(iii) and the complement of D
or A(iv)
or A(v)
A(i)
or A(ii)
or A(iii) and the complement of E
or A(iv)
or A(v)
B and the complement of D
B and the complement of E
B and the complement of F
C and the complement of D
C and the complement of E
C and the complement of F
D and the complement of E
D and the complement of F
E and the complement of F
A(i), A(ii), A(iii), A(iv), A(v), B, C, D, E and F having the nucleotide sequence indicated in Table 1.
In the case of amplification of a nucleotide sequence of the invention with any of the above mentioned primer sets defined in Table 3bis hereabove, the detection of the amplified nucleotide sequence can be achieved by a hybridization reaction with an oligonucleotide sequence of at least 10 nucleotides, said sequence being located between the PCR primers which have been used to amplify the nucleotide sequence. An oligonucleotide sequence located between said two primers can be determined from FIG. 9 where the primers A, B, C, D, E and F are represented by the boxed sequences respectively named probe region A, probe region B, probe region C, probe region D, probe region E and probe region F.
The invention also relates to a kit for enzymatic amplification of a nucleotide sequence by PCR technique and detection of the amplified nucleotide sequence containing
one of the PCR primer sets defined in Table 3 and one of the detection probes of the invention, advantageously the probes defined in Table 1, or one of the PCR primer sets defined in Table 3bis, and a detection sequence consisting for instance in an oligonucleotide sequence of at least 10 nucleotides, said sequence being located (FIG. 9) between the two PCR primers constituting the primer set which has been used for amplifying said nucleotide sequence.
The invention also relates to a process for preparing a polypeptide according to the invention comprising the following steps:
the culture in an appropriate medium of a cellular host which has previously been transformed by an appropriate vector containing a nucleic acid according to the invention,
the recovery of the polypeptide produced by the above said transformed cellular host from the above said culture medium, and
the purification of the polypeptide produced, eventually by means of immobilized metal ion affinity chromatography (IMAC).
The polypeptides of the invention can be prepared according to the classical techniques in the field of peptide synthesis.
The synthesis can be carried out in homogeneous solution or in solid phase.
For instance, the synthesis technique in homogeneous solution which can be used is the one described by Houbenweyl in the book titled ""Methode der organischen chemiexe2x80x9d (Method of organic chemistry) edited by E. Wunsh, vol. 15-I et II. THIEME, Stuttgart 1974.
The polypeptides of the invention can also be prepared according to the method described by R. D. MERRIFIELD in the article titled xe2x80x9cSolid phase peptide synthesisxe2x80x9d (J. Am. Chem. Soc., 45, 2149-2154, 1964).
The invention also relates to a process for preparing the nucleic acids according to the invention.
A suitable method for chemically preparing the single-stranded nucleic acids (containing at most 100 nucleotides of the invention) comprises the following steps:
DNA synthesis using the automatic xcex2-cyanoethyl phosphoramidite method described in Bioorganic Chemistry 4: 274-325, 1986.
In the case of single-stranded DNA, the material which is obtained at the end of the DNA synthesis can be used as such.
A suitable method for chemically preparing the double-stranded nucleic acids (containing at most 100 bp of the invention) comprises the following steps:
DNA synthesis of one sense oligonucleotide using the automatic xcex2-cyanoethyl phosphoramidite method described in Bioorganic Chemistry 4; 274-325, 1986, and DNA synthesis of one anti-sense oligonucleotide using said above-mentioned automatic xcex2-cyanoethyl phosphoramidite method,
combining the sense and anti-sense oligonucleotides by hybridization in order to form a DNA duplex,
cloning the DNA duplex obtained into a suitable plasmid vector and recovery of the DNA according to classical methods, such as restriction enzyme digestion and agarose gel electrophoresis.
A method for the chemical preparation of nucleic acids of length greater than 100 nucleotidesxe2x80x94or bp, in the case of double-stranded nucleic acidsxe2x80x94comprises the following steps:
assembling of chemically synthesized oligonucleotides, provided at their ends with different restriction sites, the sequences of which are compatible with the succession of amino acids in the natural peptide, according to the principle described in Proc. Nat. Acad. Sci. USA 80; 7461-7465, 1983,
cloning the DNA thereby obtained into a suitable plasmid vector and recovery of the desired nucleic acid according to classical methods, such as restriction enzyme digestion and agarose gel electrophoresis.
The invention also relates to antibodies themselves formed against the polypeptides according to the invention.
It goes without saying that this production is not limited to polyclonal antibodies.
It also relates to any monoclonal antibody produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat, immunized against the purified polypeptide of the invention on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by its ability to produce the monoclonal antibodies recognizing the polypeptide which has been initially used for the immunization of the animals.
The invention also relates to any antibody of the invention labeled by an appropriate label of the enzymatic, fluorescent or radioactive type.
The peptides which are advantageously used to produce antibodies, particularly monoclonal antibodies, are the following ones gathered in Table 4:
The amino acid sequences are given in the 1-letter code
Variations of the peptides listed in Table 4 are also possible depending on their intended use. For example, if the peptides are to be used to raise antisera, the peptides may be synthesized with an extra cysteine residue added. This extra cysteine residue is preferably added to the amino terminus and facilitates the coupling of the peptide to a carrier protein which is necessary to render the small peptide immunogenic. If the peptide is to be labeled for use in radioimmune assays, it may be advantageous to synthesize the protein with a tyrosine attached to either the amino or carboxyl terminus to facilitate iodination. These peptides possess therefore the primary sequence of the peptides listed in Table 4 but with additional amino acids which do not appear in the primary sequence of the protein and whose sole function is to confer the desired chemical properties to the peptides.
The invention also relates to a process for detecting in vitro antibodies related to tuberculosis in a human biological sample liable to contain them, this process comprising
contacting the biological sample with a polypeptide or a peptide according to the invention under conditions enabling an in vitro immunological reaction between said polypeptide and the antibodies which are possibly present in the biological sample and
the in vitro detection of the antigen/antibody complex which may be formed.
Preferably, the biological medium is constituted by a human serum.
The detection can be carried out according to any classical process.
By way of example a preferred method brings into play an immunoenzymatic process according to ELISA technique or immunofluorescent or radioimmunological (RIA) or the equivalent ones.
Thus the invention also relates to any polypeptide according to the invention labeled by an appropriate label of the enzymatic, fluorescent, radioactive . . . type.
Such a method for detecting in vitro antibodies related to tuberculosis comprises for instance the following steps:
deposit of determined amounts of a polypeptidic composition according to the invention in the wells of a titration microplate,
introduction into said wells of increasing dilutions of the serum to be diagnosed,
incubation of the microplate,
repeated rinsing of the microplate,
introduction into the wells of the microplate of labeled antibodies against the blood immunoglobulins,
the labeling of these antibodies being carried out by means of an enzyme which is selected from among the ones which are able to hydrolyze a substrate by modifying the absorption of the radiation of this latter at least at a given wave length,
detection by comparing with a control standard of the amount of hydrolyzed substrate.
The invention also relates to a process for detecting and identifying in vitro antigens of M. tuberculosis in a human biological sample liable to contain them, this process comprising:
contacting the biological sample with an appropriate antibody of the invention under conditions enabling an in vitro immunological reaction between said antibody and the antigens of M. tuberculosis which are possibly present in the biological sample and the in vitro detection of the antigen/antibody complex which may be formed.
Preferably, the biological medium is constituted by sputum, pleural effusion liquid, broncho-alveolar washing liquid, urine, biopsy or autopsy material.
Appropriate antibodies are advantageously monoclonal antibodies directed against the peptides which have been mentioned in Table 4.
The invention also relates to an additional method for the in vitro diagnostic of tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis comprising the following steps:
the possible previous amplification of the amount of the nucleotide sequences according to the invention, liable to be contained in a biological sample taken from said patient by means of a DNA primer set as above defined,
contacting the above mentioned biological sample with a nucleotide probe of the invention, under conditions enabling the production of an hybridization complex formed between said probe and said nucleotide sequence,
detecting the above said hybridization complex which has possibly been formed.
To carry out the in vitro diagnostic method for tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis as above defined, the following necessary or kit can be used, said necessary or kit comprising:
a determined amount of a nucleotide probe of the invention,
advantageously the appropriate medium for creating an hybridization reaction between the sequence to be detected and the above mentioned probe,
advantageously, reagents enabling the detection of the hybridization complex which has been formed between the nucleotide sequence and the probe during the hybridization reaction.
The invention also relates to an additional method for the in vitro diagnostic of tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis comprising:
contacting a biological sample taken from a patient with a polypeptide or a peptide of the invention, under conditions enabling an in vitro immunological reaction between said polypeptide or peptide and the antibodies which are possibly present in the biological sample and
the in vitro detection of the antigen/antibody complex which has possibly been formed.
To carry out the in vitro diagnostic method for tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis, the following necessary or kit can be used, said necessary or kit comprising:
a polypeptide or a peptide according to the invention,
reagents for making a medium appropriate for the immunological reaction to occur,
reagents enabling to detect the antigen/antibody complex which has been produced by the immunological reaction, said reagents possibly having a label, or being liable to be recognized by a labeled reagent, more particularly in the case where the above mentioned polypeptide or peptide is not labeled.
The invention also relates to an additional method for the in vitro diagnostic of tuberculosis in a patient liable to be infected by M. tuberculosis, comprising the following steps:
contacting the biological sample with an appropriate antibody of the invention under conditions enabling an in vitro immunological reaction between said antibody and the antigens of M. tuberculosis which are possibly present in the biological sample andxe2x80x94the in vitro detection of the antigen/antibody complex which may be formed.
Appropriate antibodies are advantageously monoclonal antibodies directed against the peptides which have been mentioned in Table 4.
To carry out the in vitro diagnostic method for tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis, the following necessary or kit can be used, said necessary or kit comprising:
an antibody of the invention,
reagents for making a medium appropriate for the immunological reaction to occur,
reagents enabling to detect the antigen/antibody complexes which have been produced by the immunological reaction, said reagent possibly having a label or being liable to be recognized by a label reagent, more particularly in the case where the above mentioned antibody is not labeled.
An advantageous kit for the diagnostic in vitro of tuberculosis comprises:
at least a suitable solid phase system, e.g. a microtiter-plate for deposition thereon of the biological sample to be diagnosed in vitro,
preparation containing one of the monoclonal antibodies of the invention,
a specific detection system for said monoclonal antibody,
appropriate buffer solutions for carrying out the immunological reaction between a test sample and said monoclonal antibody on the one hand, and the bonded monoclonal antibodies and the detection system on the other hand.
The invention also relates to a kit, as described above, also containing a preparation of one of the polypeptides or peptides of the invention, said antigen of the invention being either a standard (for quantitative determination of the antigen of M. tuberculosis which is sought) or a competitor, with respect to the antigen which is sought, for the kit to be used in a competition dosage process.
The invention also relates to an immunogenic composition comprising a polypeptide or a peptide according to the invention, in association with a pharmaceutically acceptable vehicle.
The invention also relates to a vaccine composition comprising among other immunogenic principles anyone of the polypeptides or peptides of the invention or the expression product of the invention, possibly coupled to a natural protein or to a synthetic polypeptide having a sufficient molecular weight so that the conjugate is able to induce in vivo the production of antibodies neutralizing Mycobacterium tuberculosis, or induce in vivo a cellular immune response by activating M. tuberculosis antigen-responsive T cells.
The peptides of the invention which are advantageously used as immunogenic principle have one of the following sequences:
The amino acid sequences are given in the 1-letter code.
Other characteristics and advantages of the invention will appear in the following examples and the figures illustrating the invention.